Human bone, cartilage and other tissues contain components such as hydroxyapatite, a complex calcium phosphate (Ca5(PO4)3OH) in crystalline form, and the protein collagen, which are piezoelectric; that is, which generate electric fields when mechanically stressed. In addition, the flow of fluids through vessels or intercellular spaces within tissues can also generate electric fields, so-called “streaming potentials.” In living tissue, these fields drive electric currents through the intercellular fluid creating electric potentials across the cell membranes, which may either activate receptors there directly, cause motion of ions increasing their probability of bonding to receptor sites, and/or open voltage-gated ion channels permitting motion of ions such as calcium into the intracellular medium to bond with receptors there.
Based upon research conducted in the 1960's and '70's, it appears that these electric fields are important, if not essential, to the maintenance of tissue integrity and good health, and to the repair of damage and degenerative changes which may result in whole or in part from their absence. A striking example is disuse osteoporosis, which results from limb immobilization for example while in traction or in a cast. In the absence of electric fields caused by normal use, the bone loses calcium and becomes brittle. The application of an electric field with an appropriate waveform, timing and strength from outside the body can easily prevent or reverse this form of degeneration.
Studies conducted by C. A. L. Basset, John Ryaby, Arthur Pilla and others during the 1960's and '70's identified a range of waveforms, strengths and timings with the potential to reverse osteoporosis, to accelerate the healing of broken bone, and to restart and complete the healing of nonunion fractures: those which have failed to complete healing on their own. It is believed that on the cellular level, and especially at the very small scale of processes occurring at cell membranes, these artificial signals replicate at least some of the properties of the body's own, naturally generated signals.
FIG. 1a shows the waveform induced in tissue by a pulsed electromagnetic field (PEMF) as used in early bone healing stimulators based on the work of Bassett, Pilla and others. This figure exactly reproduces FIG. 5b in U.S. Pat. No. 4,105,017, “Modification of the Growth, Repair and Maintenance Behavior of Living Tissue and Cells by a Specific and Selective Change in Electrical Environment, by John P. Ryaby and Arthur A. Pilla, issued Aug. 8, 1978. This is the waveform called “Mode 2” in that patent.
It may be noted that since this waveform represents a differential voltage, the choice of polarity shown is arbitrary and the waveform could equally well be represented by the same graph flipped top to bottom. In addition, since in PEMF the induced waveform in tissue is actually the first derivative of that in the coils, the original waveform applied to the coil is a triangle wave as shown in FIG. 1b. Since without an iron core induction at the frequencies used is quite inefficient, this triangle waveform must be applied to the coils at a power level which is orders of magnitude higher than that desired in the tissues.
The waveform comprises a sequence of pulse bursts, each nominally five milliseconds long, repeated preferably between 5 to 15 bursts per second, and separated by interburst periods of substantially no signal. Adopting the same identifying characters used in U.S. Pat. No. 4,105,017, one such burst extends from time t1 to time t5, as indicated by the brackets at the top of the figure, while the following interburst period extends from t5 to t6. A small portion of another such interburst period is shown prior to t1, while following t6, a small portion of another such burst is shown.
Within each burst of the Ryaby-Pilla “Mode 2” waveform, each individual pulse comprises three distinct phases.
A relatively long pulse, shown as positive in FIG. 1, extends from t1 to t2 with a nominal duration of about 200 microseconds. The voltage intensity within this pulse is relatively constant, with a preferred value between 1 and 3 millivolts per centimeter in the treated tissue.
This is followed by a shorter phase with the opposite polarity, shown as negative in FIG. 1, extending from t2 to t3. This phase is nominally 30 microseconds long, and may have a relatively constant intensity or one changing more or less rapidly over time, so long as the maximum intensity does not exceed about 50 millivolts per centimeter in the treated tissue.
Yet a third pulse component, containing substantially zero current, extends from t3 to t4 with a nominal duration of 10 microseconds, and represents a short “break” or “rest” at the end of each pulse before the next arrives. No mention of this third component appears in the patent claims, and Arthur Pilla has stated that it was not expected to play a role in the stimulation process. In any waveform generator driving an inductive load, as in the Ryaby-Pilla design, such a “break” may be added as a safety margin to ensure that the current from one pulse has dropped to zero before the next pulse is triggered, so successive pulses do not overlap and partly cancel each other.
Calcium-modulated protein, usually abbreviated “calmodulin” or simply “CaM,” is a small protein comprised of 148 amino acids, typically found either bound to cell membranes or free-floating in the cytoplasm near them. Its usual role is to respond to changes in the concentration of intracellular calcium ion (Ca++), which is a biochemical second messenger with many roles in the body. About twenty varieties of CaM are known, having the same overall structure but differing slightly in the details of their amino-acid sequences.
As shown schematically in FIG. 2, in the absence of Ca++, CaM normally takes a “dumbbell-shaped” conformation 100 comprising seven helical regions 102a through 102g, of which region 102d is by far the longest and forms the shaft of the dumbbell. Helical regions 102a through 102g are joined end-to-end by short flexible intermediate chains 104a through 104f. 
Of these short chains, four of them 104a, 104c, 104d and 104f (together with the ends of the helices adjoining each of them) comprise two close-set pairs of “EF-hand” regions. An EF-hand comprises about ten amino acids at the end of a first helix; a short flexible chain of twelve amino acids at least six of which have negatively-charged side chains, such as glutamate and aspartate; and about ten amino acids at the start of a second helix. When the protein molecule is undistorted, for example by external electric fields, the helices at each end of an EF-hand are approximately perpendicular to each other while the negative side chains geometrically surround a vacant center just the right size to accept and tightly bind a Ca++ ion while rejecting other small positive ions with a high degree of specificity. (Celio, Pauls and Schwaller, Guidebook to the Calcium-Binding Proteins, © 1996, Oxford University Press.) These binding sites are indicated in FIG. 2 by dashed circles 106a through 106d. 
Slight differences between their amino-acid sequences cause the Ca++ affinities and binding time constants of the four EF-hand sites to differ, so the four ions are bound sequentially and with increasing Ca++ concentration the majority of CaM may either be free or have one, two, three or all four sites filled. With each ion bound, CaM's conformation changes until when all four sites are filled by Ca++ ions 108a through 108d respectively, CaM takes instead a “horseshoe” conformation 110 with long helix 102d now separated into two portions 112a and 112b and folded like a hinge. Thus folded, the CaM molecule can wrap around and bind to a target domain 114 of any one of a large number of enzymes, thereby activating (or in some cases, deactivating) the enzyme. Ca++-CaM binding therefore lies at the start of a host of biochemical pathways within the body.
Arthur Pilla and others have proposed Ca++-CaM binding as the primary means by which electric signals within the body, when not strong enough to stimulate nerves, can still be transduced into biochemical signals. Because cytoplasm and extracellular fluids are electrically conductive while cell membranes are substantially insulating, electric fields in tissue are concentrated at and near such membranes, often exceeding the average fields in tissue by several orders of magnitude. CaM is typically located near or physically bound to such membranes.
Since CaM on the whole is approximately neutral electrically, while Ca++ is strongly charged, an electric field will cause Ca++ to move while CaM, especially if membrane-bound, remains nearly stationary. This will cause some of the Ca++ to collide with, and bind to, CaM. The PEMF waveform described in U.S. Pat. No. 4,105,017 was designed, based upon knowledge of Ca++-CaM binding kinetics, to optimize this effect causing increased binding and enzyme activation.
Since the EF-hand regions of CaM are more negatively charged than other parts of the molecule, in an electric field these regions are drawn more strongly in the direction of positive charge with resulting distortion of the molecule, as shown (again schematically) by structure 120 in FIG. 3 where all EF-hand regions 106a through 106d have been displaced in the same direction (shown in the Figure as upward) with respect to the rest of the molecule. Since helices in a protein molecule, here 102a through 102g, are relatively rigid, flexing occurs primarily in the short joining chains, with accompanying distortion of the EF-hand binding sites 106a through 106d likely impacting their ability to bind Ca++ or the kinetics or specificity of such binding. To indicate this loss or impairment of function, in FIG. 3 each binding site is indicated by a broken dashed circle rather than by a complete one as in FIG. 2.
Since CaM's primary role is to respond to changes in Ca++ concentration in the absence of electric fields, evolution appears to have favored structural variants in which optimal binding and maximum specificity for Ca++ takes place when the molecule is undistorted, while increasingly strong external fields and the resulting distortion cause increasingly less effective binding and lower specificity. The fact that the amino-acid sequence of calmodulin is strongly conserved across widely-separated species, particularly in the EF-hand regions (see for example Yang, Prokaryotic calmodulins, J. Mol. Microbiol. Biotechnol. 3(3): 457-459 (2001)), together with the geometric requirement that the helices flanking an EF-hand region be approximately perpendicular and the fact that electrical distortion of the molecule will change this angle, suggest that the molecule's sensitivity to such distortion could be quite high, with a negative impact on its functionality.
As a result, an electric field could be expected to act in either of two, mutually opposing ways: either enhancing Ca++ binding through ion motion, or discouraging it by distorting CaM binding sites making ion capture more difficult or selection less specific to Ca++. At low field intensities, with CaM still relatively undistorted, a roughly linear increase in Ca++ binding would be expected with increasing field strength. At some point, however, molecular distortion would begin to outweigh the effects of ion motion. Ca++ binding would peak, then drop off with further increases in signal strength, and ultimately fall below the level occurring with no field present.
This, in fact, is the case.
FIG. 4 illustrates typical dose-response curves for cell or tissue electrostimulation taken from two classic journal articles. (It may be noted that neither of these used a Pilla-type PEMF.) Since the amplitudes of the applied signals cover four orders of magnitude plus zero, a power-law scale was used on the horizontal axis. The vertical axis, representing the response level, is linear with the 100% level representing response of the control which had no signal applied.
Curve 150 shows ATP (adenosine triphosphate) production in response to a DC signal (Cheng et al., The effects of electric currents on ATP generation, protein synthesis and membrane transport in rat skin. Clin. Orthop. 171, 264-272 (1983)). Curve 152 shows glycine uptake in response to the same field. Both have the same form with an initial increase, passage through a maximum, then a steady decrease crossing the control level as the field strength increases, with the highest-field effects opposite to those seen at low fields. This is much the same pattern seen with most vitamins, minerals and prescribed drugs, in which small amounts are necessary for health but larger amounts can be toxic or otherwise damaging.
Curves 154 and 156 show cell response with a pulsed AC signal applied (Korenstein et al., Capacitively pulsed electric stimulation of bone cells: Induction of cAMP changes and DNA synthesis. Biochim. Biophys. Acta 803, 302-307 (1984)). For easy comparison, the signal levels of all curves have been converted to common units. Data for the AC signal were reported only for controls and at three relatively high signal levels. The dashed, low-signal portions of these curves were inferred from the slopes of lines connecting the published data points and from the fact that both responses were defined as 100% at zero signal.
Curve 154 shows cyclic adenosine monophosphate (cAMP) synthesis in response to the AC signal. cAMP synthesis is initially inhibited by stimulation, reaches a minimum, then increases until it crosses the control level and thereafter is increased. This is simply the same pattern seen in curves 150 and 152, but inverted.
Curve 156 shows DNA synthesis in response to the same AC signal. At low signal intensities it follows the same pattern as curves 150 and 152, first rising to a maximum and then declining. Upon reaching the control level, however, the curve rebounds and rises again. This suggests that there may be two different and opposing mechanisms or biochemical cascades involved, one showing stimulation and the other inhibition by the applied signal, with curve 156 representing the sum of the two. The low-signal mechanism for DNA synthesis likely involves CaM, while the high-signal mechanism may not.
Surprisingly, when all results are converted to common units of signal intensity, all of the initial deviations reach their peak values (maxima for curves 150, 152 and 156; a minimum for curve 154) at approximately the same signal level, between 10 and 100 microamperes per square centimeter. Moreover, all curves either cross or, in the case of curve 156, rebound from, the control level again at approximately the same signal level, between about 200 and about 500 microamperes per square centimeter, as indicated by dashed oval 158. This argues for a common mechanism underlying all four responses shown.
For comparison, the commonly accepted “strength-duration” or “S-D” curves as used in the present practice of electrotherapy are shown in FIG. 4b. These show the thresholds of response of various nerve types in a typical user when stimulated by isolated electric pulses of varying lengths (“durations”) and intensities (“strengths”). The curves were redrawn from Nelson, Hayes and Currier, Clinical Electrotherapy, Third Edition (Appleton & Lange, 1999) but for easy comparison with FIG. 4, the X and Y axes were reversed and the total applied current was converted to current density (the same as in FIG. 4) by assuming the stimulating signal was applied over a skin area of 100 square centimeters.
For pulses longer than about 1000 microseconds (one millisecond) the nerve response depends only on the pulse strength, while as pulses grow increasingly shorter below this limit they require correspondingly higher intensities to achieve like effects. At a signal strength below curve 160, usually nothing is felt. Such a signal is termed “subthreshold.” Above curve 160 there will normally be some sensation. Muscle stimulation begins at curve 162. At curve 164 most users begin to perceive the signal as painful, while at curve 166 most find the pain intolerable.
It is notable that the peak effects from a cell-stimulating signal fall below the sensory threshold indicated by curve 160, and thus in the subthreshold region, while the vast majority of prior art electrostimulators are intended to stimulate nerves of one type or another and thus by definition use signals whose strengths lie well above at least one of the thresholds shown. As was indicated in FIG. 4a, such strong signals are far from optimal for cell or tissue stimulation and, if used in hope of such a result, may in fact have effects opposite from those desired.
Combining Arthur Pilla's suggestion that it is electric-field-caused motion of Ca++ striking CaM which leads to increased binding and is the primary means by which relatively weak electric signals within the body are transduced into biochemical signals, with the hypothesis that CaM inherently binds Ca++ most readily and selectively when no such field is present, suggests that maximum binding will take place in response to a two-step electric process: first the application of a relatively strong field to bring Ca++ and CaM into close proximity at or near a cell membrane, then brief removal of the field so CaM can relax into its undistorted conformation for optimal Ca++ binding before the ions disperse or an oppositely-directed pulse phase draws them away again.
Depending upon its strength, the molecular environment and the Ca++ concentration, the optimal length for application of the relatively strong field may range from several tenths of a microsecond upward to several tens, hundreds or even thousands of microseconds, with an optimal value probably in the vicinity of thirty microseconds for practical field strengths in most tissues. CaM relaxation and Ca++ binding are then expected to take a further interval, again ranging from several tenths of a microsecond upward depending upon the molecular environment of the CaM and the available Ca++ concentration, with an optimal value probably in the vicinity of ten microseconds in most tissues. Only when this process is substantially complete should an electric field, directed either in the preceding direction or, usually more practically, in the opposite direction so as to create a zero-net-charge (ZNC) signal, be re-introduced.
PEMF, as stated above, stands for “pulsed electromagnetic fields” and was the original approach taken to apply weak pulsed electric fields to cells and tissues to stimulate healing, relying on electromagnetic induction to create the fields through the mechanism of eddy currents. This approach, although found safe and effective in close to a million treatment cases to date, remains grossly inefficient in terms of power since relatively large currents must flow through a coil to induce the much smaller signals actually performing the stimulation. This makes even the newest generations of PEMF devices undesirably heavy, bulky and costly.
A more direct and often preferable manner of signal delivery, using PEF or “pulsed electric fields,” was described by Kronberg in U.S. Pat. No. 5,217,009 (1993), U.S. Pat. No. 5,413,596 (1995), U.S. Pat. No. 6,011,994 (2000), U.S. Pat. No. 6,321,119 (2001), U.S. Pat. No. 6,535,767 (2003), U.S. Pat. No. 7,117,034 (2006), and published applications #2006/0293724 (2006) and #2008/0039901 (2008), all of which are hereby incorporated by reference.
With PEF, the signal in treated tissue is generated not by electromagnetic induction, but instead by applying a pulsed controlled voltage or current having substantially the same waveform as that desired in the tissues. It is applied to the target tissues through electrodes, preferably noninvasively through the skin. If signals are applied so as to set up the correct current densities in the treated tissues, taking into account their resistivities and geometry, this approach can induce the same waveforms at the cell and tissue level as in PEMF but with much greater power efficiency, at lower device cost, and often with improved user convenience.
All of the cited Kronberg patents and published applications disclose signals of the same general form, in which a continuous pulse train or a succession of pulse bursts comprises individual pulses each having just two phases, differing in length and having opposite polarities. These signals are meant to be applied to the intact human body, human tissue in vitro, an animal body or animal tissue in vitro, cultured tissue, cultured cells or other biological material, for the purpose of stimulating healing, cell or tissue repair or regeneration, cell proliferation, cell differentiation, enhanced synthesis of desired substances such as proteins, ATP, growth factors, nitric oxide or other biochemical messenger substances, or other applications as will be found by reading the cited patents.
FIG. 5, which reproduces a part of FIG. 4 in U.S. Pat. No. 5,217,009, shows a typical Kronberg waveform in a format easily compared with that of FIG. 1 in this present application. Here interval T1 corresponds exactly to Pilla's interval t1-t2, interval T2 to Pilla's interval t2-t3, interval T3 to Pilla's interval t1-t5 comprising the entire pulse burst, and interval T7 to Pilla's interval t5-t6 comprising substantially no signal. Intervals T5 and T6 at the start and end of each burst, much like Pilla's interval t3-t4, result from circuit characteristics but are not expected to have any physiological significance. Interval T4 is a charge-equalizing pulse following the burst to achieve zero net charge. Within each individual pulse there are only two intervals or phases, one positive corresponding to interval T1 and Pilla's interval t1-t2 and the other negative corresponding to interval T2 and Pilla's interval t2-t3, without any “break” or “rest” corresponding to Pilla's interval t3-t4.
Pulses comprising intervals T1 and T2 may optionally be applied instead as continuous trains not including intervals T3 through T7.
All of the cited Kronberg patents were based on the assumption that one or more conventional CMOS (complementary metal-oxide-semiconductor) logic gates or similar switching devices, in which the output must represent either a logic “1” (high) or a logic (“0” (low) voltage, would be used to form the device output. Such a device, regardless of the type of electronic switching elements actually used, appears as a switch with only two possible positions: one connected to a relatively more positive voltage level representing logic “1” or “high,” the other to a relatively more negative voltage level representing logic “0” or “low.”
For example, in a CMOS inverter, the simplest possible logic gate as shown by 170 in FIG. 6, two complementary enhancement-mode MOSFET's (metal-oxide-semiconductor field-effect transistors), a p-channel MOSFET 172 and an n-channel MOSFET 174, have the gates of both connected as an input 176 and the drain terminals of both connected as an output 178, while the source terminals of the p-channel and n-channel devices are tied to the positive supply rail 180 and the negative supply rail 182 respectively.
Applying a logic “1” or “high” to input 176 turns on the n-channel MOSFET while turning off the p-channel one, thus connecting output 178 to negative rail 182 and outputting a logic “0” or “low.” Conversely, applying a logic “0” or “LOW” to input 176 turns on the p-channel MOSFET while turning off the n-channel one, thus connecting output 178 to positive rail 180 and outputting a logic “1” or “high.” Applying an intermediate input level will turn on both the n-channel and p-channel MOSFET's weakly, thus both generating an indeterminate output (not guaranteed by the manufacturer) and wastefully allowing current to pass directly from the positive to the negative rail, so such intermediate-level input signals are normally avoided. This behavior is shown on graph 190 in FIG. 6b, in which the horizontal axis 192 indicates input voltage, curve 194 indicates the resulting output voltage, and curve 196 indicates the current.
Such a device may be conceived as a simple two-position mechanical switch 184, as shown in FIG. 7a. For clarity, the identifying characters in the following description are identical with those of corresponding parts of the MOSFET circuit which was drawn out in FIG. 6a. A movable contact 178 now forms the output, and can connect either with a positive rail 180 or a negative rail 182. Which position it takes is controlled by the voltage applied at input 176.
If two such switches 184a and 184b are connected to opposite ends of a load 186, such as a human or animal body, tissue or other biological material, as shown in FIG. 7b, the load sees a “differential” output representing the difference between discrete outputs 178a and 178b. This differential output may lie in any of three different regimes of applied voltage and resulting current.
With switch 184a “high” and switch 184b “low,” the left side of load 186 will be more positive than the right side and conventional (positive) current will flow from left to right. With switch 184a “low” and switch 184b “high,” the opposite will be true. With both switches “low,” or with both “high,” no voltage will appear across the load and no current will flow.
This third regime, with both switches alike either “high” or “low,” might initially appear to offer a simple way of implementing the “two-step electric process” described above for optimum Ca++ binding to CaM: first the application of a relatively strong field to bring Ca++ and CaM into close proximity, then brief removal of the field so CaM can return to its optimal Ca++-binding conformation before the ions disperse or an oppositely-directed pulse phase draws them away again. These two steps would be simply the application of a first- or second-regime signal—that is, either with output 178a high and output 178b low, or vice-versa—followed by a third-regime signal either with both output high or with both of them low.
On human or animal skin, however, the continued application of direct current, or of a signal containing net charge—that is, in which the integral of current over a sufficiently long time does not converge to zero—causes irritation and in time can lead to chemical burns through electrolytic changes in the skin covered by the electrodes. Analogous effects take place in the vicinity of electrodes placed in cell or tissue cultures. For this reason, zero net charge (ZNC) signals are strongly preferred in any PEF-type application.
The Kronberg patents cited above use two different but complementary approaches to achieve zero net charge. First, at the end of each pulse burst a charge-equalizing pulse, which was shown as T4 in FIG. 5, is added with timing chosen to carry substantially the same amount of charge which remains unbalanced at the end of the burst. Second, physical protection is added in the form of DC-blocking, back-to-back electrolytic capacitors. (Multiple capacitors are needed both to provide electrically symmetrical operation and, in a medical product, to assure safety in case of the failure of any single component.) The result is a modified output circuit as shown in FIG. 7c, the same as before except for the addition of capacitors 188a and 188b. 
While essential to avoid possible user injury in case of electronic failure, the DC-blocking capacitors have an undesired side effect in normal operation, in that during each pulse burst they store energy in the form of an unbalanced charge. Most of this charge is removed by the equalizing pulse following the burst, leaving a small and usually negligible amount to drain away through load 196 during the following interval of “substantially no signal” indicated by T7 in FIG. 5.
During the burst, however, a significant amount of charge builds up and this means that, if both output switches are connected to the same output rail (whether positive or negative) and thus to each other, the current in load 186 will not be zero, but instead will be determined, and powered, by the stored charge on the output capacitors as indicated by looped arrow 190. This capacitor-driven loop current will appear in the load even when third-regime (both-high or both-low) switch settings are used as portions of nominally charge-balanced signals such as those shown in U.S. Pat. No. 7,117,034, for example in its FIGS. 6 and 7, since during any third-regime interval the loop current 190 decays asymptotically toward, but never actually reaches, zero.
As a result, no signal or waveform disclosed in any of the cited Kronberg prior-art patents or other known prior art successfully implements the “two-step electric process” described above for optimum Ca++ binding to CaM. For this process to be effective, immediately following the application of current to bring Ca++ and CaM into close proximity, the current must be reduced substantially to zero, eliminating any capacitor-driven loop current, sufficiently long for CaM to return to its undistorted “natural” configuration best suited for binding the Ca++.
As a sign of the importance of this failure to implement the described “two-step electric process” in prior Kronberg patents—since at the time they were written, its importance had not yet been recognized—the long history of success of PEMF bone-healing devices using the Pilla waveform in FIG. 1, which serendipitously includes an approximation to the described “two-step electrical process,” may be contrasted with the sometimes impressive but highly inconsistent results from experimental devices based on the Kronberg patents. For example, the MedRelief SE-60, a device closely based on that described in U.S. Pat. No. 6,535,767, was tried in 2006 and 2007 by MedRelief, Inc. in an 84-subject, randomized controlled study on osteoporosis, measuring bone mineral density and biochemical markers for bone formation over eight months of PEF treatment of the spine. The PEF waveform induced in tissue by the SE-60 differs from the Pilla waveform shown in FIG. 1a chiefly through lacking Pilla's third pulse interval of substantially zero current, shown as interval t3-t4. While some patients in the study showed impressive recovery of spine mineral density, others did not, and the study as a whole failed to reach statistical significance on any of the outcome variables.
The inability to implement the described “two-step electrical process,” now thought necessary for optimal Ca++ binding to CaM initiating biochemical cascades leading to pain relief and healing, is common to the devices described in all of the cited Kronberg patents to date, due to the requirement for switching devices in which the outputs must represent a combination of logic “1” (high) and logic (“0” (low) states.
The present invention is meant to remedy this deficiency, now that it has been recognized: providing a simple yet robust and widely applicable means of implementing the “two-step electronic process” described above in any application of pulsed electric field (PEF) treatment of humans, animals, tissues, cells or other biological material.